Alkaline phosphatases (EC 3.1.3.1) are enzymes that are widely distributed from microorganisms to humans and hydrolyze phosphoric monoesters to produce inorganic phosphoric acids. Alkaline phosphatases are generally known to be metal-dependent enzymes that have low substrate specificity and require metal ions such as magnesium ions (Mg2+) or zinc ions (Zn2+) for enzymatic reactions. Typical alkaline phosphatases include bacterial alkaline phosphatase (BAP), calf intestinal alkaline phosphatase (CIAP), shrimp alkaline phosphatase (SAP) and the like. Particularly, alkaline phosphatases are industrial enzymes which are widely used in the industrial fields, including the molecular biology field. Alkaline phosphatases are used in genetic manipulations such as cloning. Specifically, alkaline phosphatase is used to remove a phosphoric acid molecule from the ends of a vector in order to prevent self-ligation of the vector after the vector is digested with restriction enzymes for an insertion of a target gene. In addition, alkaline phosphatase is used as a typical reporter protein together with peroxidase in antibody-based immunoassays such as enzyme-linked immunosorbent assay or Western blotting, and otherwise it is also used to measure the effect of milk pasteurization. Enzymes having activities similar to alkaline phosphatase include phytase and pyrophosphatase. Phytase is an industrially highly valuable enzyme which is used for feed and environmental treatment, and pyrophosphatase is used to increase the efficiency of polymerase chain reaction (PCR).
Meanwhile, Handelsman et al. define a metagenome as collective genomes of all microorganisms in a given habit (Handelsman et al., (1998) Chem. Biol. 5: R245-R249). However, in recent years, the term “metagenome” refers to a collection of clones, including genomes or genes extracted from environmental samples, and a series of studies related to the metagenome are also defined as metagenomics (Chen et al., (2005) PLoS Comput. Biol. 1(2): 24). In studies related to the metagenome, Venter et al. (Venter et al., (2004) Science 304: 66-74) conducted a new conceptual study (entitled “Ecosystem sequencing”) under the support of the US Department of Energy (DOE) and determined about 1 billion nucleotide sequences by whole-genome shotgun sequencing of a metagenomic library constructed from seawater samples collected from the Bermuda Triangle. The results of the shotgun sequencing indicated that the collected seawater samples contained at least 1800 microbial genomic species, including 140 unknown phylotype bacteria, and 12 millions or more new genes were found. Metagenomic studies are well suited for the purpose of using microorganisms which are difficult to culture or impossible to culture, and such metagenomic studies are molecular biology approaches which comprise extracting all DNAs in a specific environment if the microorganisms of interest are impossible to isolate by culture.
In recent years, studies on the use of directed molecular evolution technology to ensure new genetic sources from microbial genomes or metagenomes or improve the activities of existing genes to develop highly useful biocatalysts have received attention as an important strategy of biotechnology. Thus, there have been attempts to screen various enzymes using new high-sensitivity screening technologies of rapidly sensing the activities of very small amounts of enzymes.
Methods of selecting new enzymatic genes from massive gene libraries such as microbial genomes or metagenomes include a sequence-based screening method comprising performing polymerase chain reaction (PCR) based on DNA nucleotide sequences and selecting the amplified genes. This method has advantages in that the utility thereof increases day by day as genomic information increases rapidly and in that only desired genes can be specifically screened. However, this method has a disadvantage in that subjects to which the method can be applied are limited to some genetic sources, because the method can be used only in the case in which accurate information on the nucleotide sequence of a target gene is known.
In addition to this method, a function-based screening method of screening genes based on the genetic functions (i.e., enzymatic activities) is also widely used. This method has an advantage in that it can screen new unknown genes based on the enzymatic activities. However, this method has a limitation in that a screened strain should have a cluster of all activity-related genes in order to have activity and should be able to be expressed in a library host strain (e.g., E. coli).
In this context, studies on genetic circuitries which sense the activity of enzymes such as intracellular protease using genetic engineering technology based on the principle of transcriptional activity of yeast 3-hybride have received attention. Furthermore, there have been active studies on the technology of detecting enzymatic activity using microbial metabolic pathways designed such that metabolic products of foreign enzymes provide nutrient sources to cells. Also, technology of introducing a transcription regulatory protein, derived from microorganisms, into recombinant E. coli and sensing the enzymatic activity of the foreign gene in the recombinant E. coli has been actively studied. In addition, an effort has been actively made to develop protein engineering technology for improving the substrate specificity of regulatory proteins. For example, in one study, the regulatory protein HbpR that binds to 2-hydroxybiphenyl (2-HBP) was improved such that it specifically recognizes 2-chlorobiphenyl (2-CBP) having a chloro group in place of a hydroxyl group (Beggah et al., (2008) Microb. Biotechnol. 1(1): 68-78). In particular, studies on screening new enzymes using genetic circuitries that sense reaction products based on regulatory proteins have received a great deal of attention as a new technology for screening enzymatic activities.
Accordingly, the present inventors have made extensive efforts to screen new active enzymes, derived from metagenomes, by detecting various enzymatic activities with high sensitivity, and as a result, have constructed an artificial genetic circuit capable of detecting various phenolic compounds, and recovered and purified a novel gene having alkaline phosphatase enzymatic activity from a metagenomic library by screening using the genetic circuit in a high-throughput manner, thereby completing the present invention.